A Double-Locked ESIPT-AIE Fluorescent Probe Detects Esterase with Highly Matched Response Kinetics

Hydrolyases play an irreplaceable role in complex biological processes, and their dysfunction is a cause of many human diseases. Advanced activatable in situ fluorescence detection methods offer high-resolution spatiotemporal analysis, aiding in the dissection of the complex biological roles of hydrolases. However, current strategies typically focus on only specific stages of enzyme-probe interactions, leading to suboptimal imaging fidelity and sometimes erroneous detection results. Addressing this, we developed a double-locked "Excited State Intramolecular Proton Transfer-Aggregation Induced Emission (ESIPT-AIE)" fluorescent probe (Br-3N-2Et) that matches the entire enzymatic response kinetics for enzyme activity detection. We validated the probe's mechanism by enhancing pre-reaction recognition through double unlockable recognition sites, thereby reducing basal fluorescence (Φ = 0.0183) and increasing resistance to interference signals. Subsequently, the ESIPT fluorophore with multiple hydrogen bonds enhanced the affinity for the hydrolase catalytic site, improving binding kinetics and exhibiting a significant Stokes shift (188 nm). The realization of the ESIPT-AIE dual-emission mechanism facilitated rapid efflux of the fluorophore from the catalytic site and subsequent in situ fluorescence signal enhancement (132.2-fold). This new probe achieved regional differential detection of esterase activity in HepG2 cells and endometrial cancer tissues. Thus, this work paves the way for the development of integrated, multimechanism platforms for hydrolase activity fluorescence sensing and imaging in complex biochemical contexts.